View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central The Desmoplakin Carboxyl Terminus Coaligns with and Specifically Disrupts Intermediate Filament Networks When Expressed in Cultured Cells Thaddeus S. Stappenbeck and Kathleen J. Green Department of Pathology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 Abstract. Specific interactions between desmoplakins tides including the 90-kD carboxy-terminal globular I and 11 (DP I and II) and other desmosomal or cyto- domain of DP I specifically colocalized with and ulti- skeletal molecules have been difficult to determine in mately resulted in the complete disruption of IF in part because of the complexity and insolubility of the both cell lines. This effect was specific for IF as micro- desmosome and its constituents . We have used a mo- tubule and microfilament networks were unaltered . lecular genetic approach to investigate the role that This effect was also specific for the carboxyl terminus DP I and 11 may play in the association of the desmo- of DP, as the expression of the 95-kD rod domain of somal plaque with cytoplasmic intermediate filaments DP I did not visibly alter IF networks. Immunogold (IF) . A series of mammalian expression vectors en- localization of COS-7 cells transfected with constructs coding specific predicted domains of DP I were tran- including the carboxyl terminus of DP demonstrated siently expressed in cultured cells that form (COS-7) an accumulation of mutant protein in perinuclear aggre- and do not form (NIH-3T3) desmosomes. Sequence gates within which IF subunits were sequestered. encoding a small antigenic peptide was added to the 3' These results suggest a role for the DP carboxyl ter- end of each mutant DP cDNA to facilitate immuno- minus in the attachment of IF to the desmosome in ei- localization of mutant DP protein. Light and electron ther a direct or indirect manner. microscopical observations revealed that DP polypep- DMOSOMEs are intercellular junctions that function Ultrastructurally, desmosomes appear as disc-shaped in cell-cell adhesion and act as specific cell surface regions, 0.5-2.0 Am in diameter and -100-run thick, sym- attachment sites for intermediate filaments (IF), metrically arranged about two adjacent cell membranes. Be- (for reviews see Steinberg et al., 1987; Garrod et al., 1990; tween the plasma membranes is a 20-30-nn space which Green andJones, 1990; Schwarz et al., 1990). The supracel- contains the extracellular domains of the transmembrane lular network of cytoplasmic IF and desmosomes is found glycoproteins of the desmosome (or desmoglea). The cyto- in a number of tissues such as epithelia, myocardium, and plasmic portion of the desmosome consists of an electron- arachnoid where desmosomes interact with keratin, desmin, dense, trilaminar plaque of variable thickness (10-40 nn), and vimentin IF, respectively (Schwarz et al., 1990). While which underlies both cell membranes (Schwarz et al., 1990). the exact function of IF has yet to be fully understood IF are thought to interact with the desmosome by looping (Steinert and Roop, 1988 ; Klymkowsky et al., 1989), the ul- through the innermost plaque region (Kelly, 1966). trastructure of the IF-desmosome network suggests that in The highly related molecules, desmoplakins I and II (DP addition to maintaining the integrity and proper relationship I and II), are the most abundant constituents ofthe cytoplas- among cells, it may impart increased tensile strength and me- mic plaque region of the desmosome (Mueller and Franke, chanical resistance to whole tissues (Arnn and Staehelin, 1983; Kapprell et al ., 1985). Based on an analysis of the 1981). Recent work that supports this idea demonstrated that predicted amino acid sequence (Green et al., 1990), DP I is transgenic mice with disrupted IF in the basal layer of strat- predicted to form a homodimer comprising a central «-he- ified epithelia displayed abnormalities in epidermal architec- lical coiled-coil dimer -130-nn long, flanked by two globu- ture and blistered easily (Vasser et al., 1991). Although cy- lar ends corresponding to the amino and carboxy termini of toplasmic IF have been extensively investigated, the exact themolecule. This prediction is consistent with the biochem- nature and importance of the link to desmosomes is un- ical evidence that purified and crosslinked DP I exists as a known. dimer in vitro. Furthermore, rotary shadowed images of 1. Abbreviations used in this paper: DP, desmoplakins ; IF, intermediate fila- purified DP I (OKeefe et al., 1989) appeared dumbbell ments; PCR, polymerase chain reaction. shaped with a central "rod" of the length predicted by Green © The Rockefeller University Press, 0021-9525/92/03/1197/13 $2 .00 The Journal of Cell Biology, Volume 116, Number5, March 1992 1197-1209 1197 et al. (1990). The rod domain of DP I is characteristic of Construction ofExpression Vectors many a-fibrous proteins including IF in that it is predomi- For each construct, polymerase chain reaction (PCR) mutagenesis was per- nantly composed of a series of heptad repeats (Conway and formed to generate sequence for a molecular tag, an artificial start, and a Parry, 1990). DP II is thought to be derived from an alterna- stop site. AmpliIàq DNApolymerase (Perkin-Elmer Corp., Norwalk, CT) tively spliced mRNA of DP I resulting in a greatly shortened was used according to the manufacturer's instructions in a reaction with 10 rod domain (Green et al., 1990). ng of the appropriate DP I cDNA and 1 gM of the appropriate oligos. After PCR amplification for 35 cycles (1 min at 94°C, 2 min at 45°C for cycles One particularly interesting feature of the 851 amino acid 1-5, and 2 min at 55°C for cycles 6-35, 1 min at 72°C), products were iso- carboxyl terminus of DP I and II is a series of three regions lated from a polyacrylamide gel, subcloned into pGEM-9Zf(-) (Promega that contain 4.6 copies of a 38-residue repeat (Green et al., Corp., Madison, WI) and sequenced using the Sequenase Kit (United States 1990). The periodicity of the acidic and basic residues of Biochemical, Cleveland, OH) to confirm that the correct sequences had been inserted . these repeats matches that of the 1B rod domain of IF thus Construction of pDPCT. A 34-mer oligonucleotide 5'-AAGAGCT indicating a basis for potential ionic interaction between the CGCCATGGgagcatctgcttctccta-3' (Northwestern Biotechnology Research two molecules . The possibility that DP links IF to the des- Service Facilities, Chicago, IL) was used to insert sequence encoding a Sacl mosome has been previously suggested based on a number restriction site followed by a portion of the Kozak (Kozak, 1986) consensus sequence for ribosome binding and an ATG start site (both underlined) 5' of observations (Green and Jones, 1990). Furthermore, the of the predicted carboxyl terminus (lower case). This oligo in the sense 38-residue repeat has also been found in the carboxyl termi- orientation was used in a PCR reaction with an antisense oligo 5'-CTGT nus of plectin, which is a known IF-associated protein CGACAGTCAGCTT 3' that was located at an internal Sall site 150-bp (Wiche et al., 1991), and the 230-kD bullous pemphigoid an- downstream from the start site of the carboxyl terminus . This 150-bp tigen (Green et al., 1990; Tanaka et al., 1991), which has SacI/Sall fragment was subcloned into a plasmid containing a 2 .4-kb SaII/EcoRI fragment ofDP cDNA encoding a majorportion ofthe carboxyl been localized to the plaque region of hemidesmosomes. terminus (pl) . A 71-mer oligo 5'-agcagtagttctattgggcac GTGGAGCAAAAG- Hemidesmosomes act as attachment sites for IF at the CTCATTTCTGAAGAGGACTTGTAGGGTACCGAATTCCC-3' was used dermal-epidermal junction (Green and Jones, 1990), so it is to insert sequence encoding a 33-bp c-myc epitope (underlined), a stop intriguing that this junction contains a plaque component codon and a KpnI and an EcoRl restriction site at the 3' end of the DP I similar to DP in desmosomes. cDNA (lower case). This oligo in the antisense orientation was used in a PCR reaction with a sense oligo 5'-GTAGGAAGAATTCCTGC-3' that was One difficulty with the proposed DRmediated linkage of located 70-bp upstream from the end of DP I at an EcoRl site . The EcoRI IF to the desmosome is that previous attempts using bio- fragment was subcloned into pl. The full DPCT was then cut out with a chemical techniques have been unable to demonstrate an in- SacI/KpnI digest and blunt end ligated into pRC4B (kindly provided by Dr. teraction between DP and IF or any other molecule (O Keefe R. Scarpulla, Northwestern University, Evanston, IL) . Construction of pDPROD. A 35-mer oligonucleotide, 5'-AAGAGCT et al ., 1989 ; Pasdar et al ., 1991) . There are several possible CGCCATGGagaaagccatcaaggagaag-3' was used to insert sequence encoding explanations for these results. First, ifDP does directly bind a SacI restriction site followed by aportion of the Kozak consensus sequence IF, it is possible that the denaturing conditions used to ex- for ribosome binding and an ATG start site (both underlined) 5' of the tract DP from desmosomes during purification affected the predicted rod domain (lower case). A 49-mer oligonucleotide, 5'-TTGAGC- in binding capacity of DP Another possibility is that TCCTACATGAGGCCGAAGAAagcgatagatcctgcaccccgaa-3' was used to in- vitro sert sequence encoding a 15-bp fragment of the neuropeptide substance P DP may interact directly with IF, but only in the presence (underlined), a stop codon and a SacI restriction site 3' of the predicted end of accessory proteins that would stabilize the interaction. A of the rod domain (lower case). These twooligos (the former sense, the lat- third possibility is that one or more linking proteins mediate ter antisense) were used in a PCR reaction to generate a 2.7-kb Sacl frag- an indirect association of DP and IF.